Vegetal diamine oxidase alleviates histamine-induced contraction of colonic muscles

Abstract

Excess of histamine in gut lumen generates a pronounced gastrointestinal discomfort, which may include diarrhea and peristalsis dysfunctions. Deleterious effects of histamine can be alleviated with antihistamine drugs targeting histamine receptors. However, many antihistamine agents come with various undesirable side effects. Vegetal diamine oxidase (vDAO) might be a relevant alternative owing to its histaminase activity. Mammalian intestinal mucosa contains an endogenous DAO, yet possessing lower activity compared to that of vDAO preparation. Moreover, in several pathological conditions such as inflammatory bowel disease and irritable bowel syndrome, this endogenous DAO enzyme can be lost or inactivated. Here, we tested the therapeutic potential of vDAO by focusing on the well-known effect of histamine on gut motility. Using ex vivo and in vitro assays, we found that vDAO is more potent than commercial anti-histamine drugs at inhibiting histamine-induced contraction of murine distal colon muscles. We also identified pyridoxal 5'-phosphate (the biologically active form of vitamin B6) as an effective enhancer of vDAO antispasmodic activity. Furthermore, we discovered that rectally administered vDAO can be retained on gut mucosa and remain active. These observations make administration of vDAO in the gut lumen a valid alternative treatment for histamine-induced intestinal dysfunctions.

Figure 1

Assay of DAO activity from various sources by spectrofluorimetry. Specific activity expressed in units (U)/mg protein of DAO from (A) homogenates of various intestinal segments of mice and from (B) vegetal (Lathyrus sativus) and animal (pig kidney) sources in the absence (black) or presence (white) of semicarbazide (mean ± SD; n = 3; ** P < 0.01, *** P < 0.001; one-way ANOVA multiple comparisons).

Figure 2

Time course of histamine-triggered distal colon muscle contraction in absence or in presence of vDAO ex vivo. Upper panels are representative contractility curves induced by 50 µM histamine (A) alone or (C) in presence of vDAO 1.25 mg/mL. Middle panels are quantitative analyses of contractility (expressed in g/s) corresponding to the difference in the area under the curve (ΔAUC) after and before addition of various concentrations of (B) histamine or (D) vDAO and 50 µM histamine. Lower panels are control experiments showing that oxidative deamination of histamine is slower at lowest concentration of vDAO (E), and that the vDAO preparation has no impact on histamine-induced contractions when inactivated with the specific Cu-AO inhibitor (topaquinone-binding) semicarbazide (F) (n = 3–4; * P < 0.05, ** P < 0.01, *** P < 0.001; one-way ANOVA multiple comparisons).

Figure 3

Effect of commercial antihistamine agents on histamine-triggered distal colon muscle contraction ex vivo. The effect of desloratadine (Deslo) antihistamine drug on the ex vivo histamine-induced contractile response (Hist 50 µM, left side) and the related quantitative analysis (right side) in the presence of (A,B) desloratadine or of (C,D) pig kidney (pk) DAO at various concentrations (n = 3; ** P < 0.01, *** P < 0.001; one-way ANOVA multiple comparisons).

Figure 4

Effect of the H₂O₂ by-product of vDAO reaction on distal colon muscle contraction ex vivo. (A) Determination of H₂O₂ released in vitro by 5 mg solid/mL vDAO in presence of 50 µM histamine, using a H₂O₂ standard curve (inset in A). (B) Ex vivo effects of varying concentrations of H₂O₂ on muscle contraction (n = 3; *** P < 0.001; one-way ANOVA multiple comparisons).

Figure 5

PLP enhances the antispasmodic effect of vDAO ex vivo. Representative contractility curves (left panels) and corresponding quantitative analyses (right panels) for (A,B) PLP alone, or in presence of (C,D) Histamine and of (E,F) Histamine and vDAO (n = 3; ** P < 0.01, *** P < 0.001; one-way ANOVA multiple comparisons).

Figure 6

In vitro analysis of vDAO activity as a function of PLP concentration. (A) Representative zymography gel and (B) corresponding densitometry analysis of vDAO activity at different concentrations of PLP. (C) Coomassie staining of zymography gel indicating equal loading and appropriate molecular weight of vDAO monomer (75 kDa) according to Broad range SDS-PAGE molecular weight standards (std). (D) Ammonia concentration released during the vDAO activity in presence of various concentrations of PLP (values are mean ± SD; n = 3).

Figure 7

Retention of active vDAO on colon mucosa after rectal administration. (A) Analysis of vDAO-mediated release of H₂O₂ from colon mucosa of mice that previously received vDAO by enema. (B) Analysis of vDAO retention by staining with FITC-labeled Concanavalin A. (C) Retained vDAO remains active after repeated washes (mean ± SD; n = 3–5; * P < 0.05, ** P < 0.01; one-way ANOVA multiple comparisons).